High resolution optical imaging is an important tool in many fields of physical science, and especially in biology and medicine. Far field optical microscopy techniques are used extensively for imaging biological samples with diffraction limited resolution of ˜300 nm. Near field applications, such as optical data storage, inspection, microscopy, allows imaging with resolution below the optical diffraction limit by generating a point-like light source of sub-wavelength dimensions nearby the sample surface. This is typically achieved either by defining small apertures on opaque screens, or by passing the light through point-like tips of sub-wavelength dimensions. The tips (constituting point-like light sources) are located in close proximity of the object (the sample surface) in order to provide high optical resolution of the scanning system in the near field.
Near field scanning optical systems often utilize the methods employed in widely spread scanning probe microscopy (SPM) techniques. Among these techniques, scanning tunneling microscopy (STM) for studying conductive surfaces and atomic force microscopy (AFM) for studying also non-conductive surfaces, are the most wide-spread techniques. The AFM methods are of particular relevance and are based on the principle of force sensing between a tip proximal to the sample surface. More specifically, a sharp point is fixed to the end of a spring-like cantilever and is brought so close to the surface that the forces between the tip and the surface deflect the cantilever. This deflection is detected most commonly by means of sensing the position of a light beam reflected from the cantilever onto a split photodiode detector. In one common AFM mode, contact mode, the measured deflection is translated into a correction signal that is used as feedback to keep the deflection constant by moving the cantilever up or down and thus reflecting the sample surface topography. Other methods are known for AFM including tapping mode AFM and conductive AFM.
The resolution of near field scanning optical microscopy (NSOM) obtainable with conventional tapered fiber probes is typically on the order of 100 nm. It is difficult to improve the resolution of this technique to the molecular level due to the finite skin depth of the metal coating surrounding the fiber probe, and its low throughput and low damage threshold. This limitation can be overcome by implementing apertureless-NSOM techniques. The contrast mechanism of these methods is based on detecting near field effects, locally induced by a sharp probe proximal to the sample. With the increasingly wide-spread and robust implementation of AFM (atomic force microscopy) schemes briefly described above, the aperturelss-NSOM techniques also become more accessible.
One approach to enhance optical resolution via aperturless-NSOM is the exploitation of strongly distance dependant physical interactions such as FRET (fluorescence resonance energy transfer) [1,2]. FRET is widely used in solution experiments and in single molecule spectroscopy, to determine molecular scale distances in biological samples. The intensity of the FRET signal scales as the inverse sixth power of the distance between donor and acceptor molecules [3]. The range of the FRET process can be estimated from R0, the distance where the interaction is at 50% efficiency, with typical values of 1-10 nm. During the FRET process, energy is transferred non-radiatively through a dipole-dipole interaction from the excited donor chromophore, to the acceptor which fluoresces. Detection of the relative intensities of donor and acceptor fluorescence provides information regarding their relative distance and orientation [4, 5]. This high sensitivity of FRET to molecular scale distances has been suggested as a contrast mechanism for high resolution optical imaging [2].
FRET based microscopy schemes are realized by the immobilization of donor or acceptor chromophores on the tip of a scanning probe microscope used to image the complimentary FRET species on the substrate. As the functionalized tip approaches a chromophore on the substrate, the FRET interaction leads to donor quenching while inducing acceptor emission, indicating the position of the chromophore with potential for molecular-scale resolution.
Several attempts to realize this imaging technique have been reported using pairs of dye molecules. For example, Shubeita et al [6] coated NSOM tips with polymer containing acceptor molecules.
Semiconductor nanocrystals have several advantages over dye molecules as FRET donors. These advantages have also prompted their emerging use as novel biological markers in both in vitro and in in-vivo applications. First, the nanocrystals may be tailored, via control of size, composition and shape [7] to provide exceptional spectral coverage with symmetric emission profiles, enabling optimization of donor-acceptor spectral overlap. Additionally, due to their continuous absorption band they may be excited efficiently at shorter wavelength regions where the acceptor dye molecule has minimal absorption cross section reducing direct acceptor excitation and hence donor-acceptor cross-talk. Finally, as already demonstrated in several applications [8], the nanocrystals are significantly more stable emitters compared to the conventional dye molecules and as mentioned above, this is a critical feature for a feasible FRET microscopy scheme.
Recently, CdSe—ZnS quantum-dots were used as FRET donors in a model protein-protein binding assay demonstrating their advantages for FRET applications [9]. In addition, Shubeita et al [10] have recently used semiconductor nanocrystals to coat NSOM fiber tips. In that case, the fiber tips were dipped in a polymer solution containing the nanocrystals to yield a 30-100 nm thick layer of nanocrystal-stained polymer. The polymer was used to embed the nanoparticles on the fiber tip.
A metal coated AFM tip where the coating was deposited by sputtering was shown by Anderson [16] to yield local enhanced Raman signal.